Method of effecting neuroprotection using soluble neuregulin isoforms

ABSTRACT

The present invention refers to soluble Neuregulin-1 isoforms representing Posttranslational Neuregulin-1 modifications as medication in cognition-related neurological disorders, in particular schizophrenia, Alzheimer&#39;s and Parkinson&#39;s diseases.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/012,159, filed Sep. 4, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/420,755, filed May 23, 2019, now U.S. Pat. No.10,813,978, which is a division of U.S. patent application Ser. No.15/333,959, filed Oct. 25, 2016, now U.S. Pat. No. 10,350,269, which isa continuation of U.S. patent application Ser. No. 14/271,617, filed May7, 2014, which is a division of U.S. patent application Ser. No.12/742,983, filed May 14, 2010, which is a 35 U.S.C. 371 National PhaseEntry Application from PCT/EP2008/009715, filed Nov. 17, 2008, whichclaims priority from and the benefit of U.S. Ser. No. 60/988,576 filedon Nov. 16, 2007, each of which are incorporated herein in theirentireties by reference.

DESCRIPTION

The present invention refers to Neuregulin-1 isoforms soluble inphysiological solutions representing posttranslational Neuregulin-1modifications or splice variants as medication in cognition-relatedneurological disorders, in particular schizophrenia, Alzheimer's andParkinson's diseases.

BACKGROUND OF THE INVENTION

Neuregulins (NRG) have emerged as key regulators of synaptic signalling.These transmembrane proteins are encoded by four genes (NRG-1, -2, -3and -4), and their diversity is further increased by alternate RNAsplicing and promoter usage and in particular by posttranslationalmodifications like proteolytic processing which leads to release ofsoluble isoforms from membrane-bound holoproteins. Moreover there isevidence of phosphorylation and glycosylation (Buonanno and Fischbach2001). They are characterized by different extracellular domains and areligands of ErbB receptor tyrosine kinases, which have downstreamconnotations to neuroinflammation and gene transcription (Holbro andHynes 2004). In particular, soluble isoforms of NRG-1 are produced fromthe transmembrane form of NRG through proteolytic cleavage duringelectrical stimulation and subsequently secreted as activity-dependentsynaptic modulators (Ozaki et al. 2004).

A truncated isoform of NRG-1, presumably β1, comprising the N-terminalextracellular domain (ECD) of the entire membrane protein, which hasbeen found to be correlated to learning and memory (Schillo et al.2005a; WO03/014156). Functional studies have demonstrated, that NRG-1directly regulates NMDA receptor subunit composition (Ozaki et al. 1997;Eilam et al. 1998). Moreover it has been shown that NRG-1 fragments ofthis type have neuroprotective properties in vivo by antiapoptoticeffects (Xu et al. 2005A; Xu et al. 2005B; Xu et al. 2004).

Very recently it became clear that NRG-1 has a central role in humanneurological diseases due to NRG-dependent regulation of NMDA receptors(Schrattenholz and Soskic 2006), and subsequent downstream events likeexcitotoxicity, neuroinflammation and apoptosis (see FIG. 1 forsummary). There are results showing that NRG 1 plays a pivotal role inconditions ranging from amyotrophic lateral sclerosis, Alzheimer's andParkinson' disease, to stroke and schizophrenia (Britsch 2007).

This fundamental significance of NRG-1 implies that next toneuroprotection and a positive role in cognition-related learning andmemory, NRG-1 represents a crucial neurotrophic factor in regenerationof neuronal tissue after a variety of lesions, in a variety of specificbrain regions and cell types. Obviously it is the crucial factor formaintenance and repair of the integrity of neuronal circuitry:neuroprotective and with roles in correct regeneration after loss offunction, as well as in the formation of activity-dependent neuronalplasticity.

The interest in Neuregulin 1 β was further fueled considerably whenKastin et al., 2004, showed that Neuregulin 1 β is able to cross theblood-brain barrier. That opened the perspective for the therapeuticusage of Neuregulin 1 β.

Latest research proved the breadth of application in neuroprotection.Independently it was shown in two publications that Neuregulin 1 is alsoa substrate of BACE (β-secretase, β-amyloid converting enzyme), whichindicates the relevance of Neuregulin 1 in Alzheimer's disease (Glabe2006; Schubert 2006).

Further, it was found that in Schwann cells neuregulin-1 increases thetranscription of the 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase,the rate-limiting enzyme for cholesterol biosynthesis in Schwann cells(Pertusa et al. 2007). This has far reaching implications for allconditions where the myelin sheath is affected, e.g. schizophrenia andmultiple sclerosis, or cognition-related functions, where so-called“cholesterol-rich rafts” are involved (Schrattenholz and Soskic 2006).Schwann cell surrounding axons express NRG1 (receptors ErbB2/ErbB3 andsoluble NRG1 α and β under physiological conditions Followingdenervation, adult Schwann cells leave the contact with axon, changetheir morphology, stop expressing NRG1β, and upregulate NRG1 α andErbB2/ErbB3 expression (Gewia et al, 2007 Karoutzou et al. 2007).

In addition, genetic epidemiologic research shows the clear associationof Neuregulin 1 to schizophrenia and to Alzheimer disease, and inparticular to its psychotic forms (Farmer et al., 2007).

Some recent genetic population analyses show, that certain NRG-1-SNP'sare associated with Alzheimer and schizophrenia (Go et al. 2005;Scolnicket al. 2006;Ross et al. 2006;Meeks et al. 2006;Farmer et al. 2007). Theimplications of these findings are related to other proteins of thefunctional NRG-containing complex depicted in FIG. 1 (ErbB receptor:(Benzel et al. 2007;Thomson et al. 2007;Hahn et al. 2006). There is alsoan implication for NRG-1 in multiple sclerosis (Esper et al. 2006).

There are results suggesting that the molecular mechanism of theassociation between NRG1 risk alleles and schizophrenia may includedown-regulation of nicotinic acetylcholine receptors of alpha7subtype(Mathew et al. 2007).

According to the present invention it was found that recombinant solubleNeuregulin-1 β isoforms show pharamceutical efficacy in animal modelsfor learning and memory, schizophrenia, Alzheimer's disease andParkinson's disease. After i.v. administration, Neuregulin-1 β isoformswere active at concentrations which are significantly lower thanconcentrations of control medicaments.

Thus, a first aspect of the present invention is the use of arecombinant soluble Neuregulin-1 isoform for the manufacture of amedicament for the treatment of neurological conditions, particularly ofcognition-related neurological conditions.

A further aspect of the present invention is a pharmaceuticalcomposition or kit comprising (i) a recombinant soluble Neuregulin-1isoform and (ii) a further medicament particularly for the treatment ofneurological conditions, particularly of cognition-related neurologicalconditions.

Still a further aspect of the present invention is the use of arecombinant soluble Neuregulin-1 isoform for memory and cognitionenhancement for the manufacture of a medicament.

Still a further aspect of the present invention is a method of treatinga neurological condition comprising administering a recombinant solubleNeuregulin-1 isoform in a pharmaceutically effective amount to a subjectin need thereof.

Still a further aspect of the present invention is a method forenhancing memory and cognition comprising administering a recombinantsoluble Neuregulin-1 isoform in a pharmaceutically effective amount to asubject in need thereof.

Still a further aspect of the present invention is a co-administrationof a recombinant soluble Neuregulin-1 isoform together with a furthermedicament.

According to the present invention, soluble Neuregulin-1 isoforms havebeen found to be effective for the treatment of neurological conditions,particularly conditions, such as psychotic disorders like schizophrenia,bipolar disorder and depression, neurodegenerative disorders, likeParkinson's disease, Alzheimer's disease, Multiple Sclerosis (MS), orAmylotrophic Lateral Sclerosis (ALS), epilepsy or neurological injurylike stroke, traumatic brain injury and spinal chord injury. Preferredis the treatment of schizophrenia, in particular cognition-relatedaspects of schizophrenia, Parkinson's disease and Alzheimer's disease.Further, the invention also refers to the use of recombinant solubleNeuregulin-1 isoforms for memory and cognition enhancement, particularlyfor reducing and/or inhibiting memory and cognition loss associated witha neurological condition such as Alzheimer's disease and schizophrenia.

The recombinant soluble Neuregulin-1 isoform is preferably a humanNeuregulin-1 isoform, i.e. a recombinant isoform comprising the primaryamino acid sequence of a naturally occurring human Neuregulin-1 isoformor a sequence which has a identity of at least 90%, preferably at least95% and most preferably of at least 98% based on the total length of therecombinant isoform.

The soluble recombinant Neuregulin-1 isoform of the present inventionpreferably comprises at least a portion of the extracellular domain ofthe corresponding Neuregulin-1, e.g. at least a portion of theextracellular domain of a human Neuregulin, e.g. human Neuregulin-1 β.

The recombinant soluble Neuregulin isoform of the present inventionpreferably has a length of up to 250 amino acids, e.g. 150 to 250 aminoacids. The molecular weight of the Neuregulin isoform is preferably ofabout 15 to about 35 KD, particularly about 25 to about 32 KD, asmeasured e.g. by SDS-polyacrylamide electrophoresis (PAGE). Therecombinant soluble Neuregulin-1 isoform, particularly the recombinantNeuregulin-1 β isoform, has an isoelectric point (pI) of about 4 toabout 9.5, preferably of about 4 to about 6. The isoform may be anunmodified polypeptide which consists of an unmodified amino acidsequence or a modified polypeptide, wherein the modification may beselected from phosphorylation, glycosylation, methylation,myristylation, oxidation and any combination thereof. In an especiallypreferred embodiment, the Neuregulin-1 isoform comprises at least onephosphorylated amino acid residue. Further, the present inventionencompasses conjugation to heterologous moieties such aspoly(alkyleneoxide) moieties, particularly polyethylene glycol moieties.

The recombinant soluble isoforms may be administered according to anyroute by which effective delivery into the target tissue, e.g. thenervous system, particulary the central nervous system, such as brainand/or spinal chord, is achieved. It was found that pharmaceuticallyeffective concentrations of Neuregulin isoforms may be achieved bysystemic administration. For example, the isoforms may be administeredby injection or infusion, e.g. by intravenous injection. The isoformsare preferably administered in an amount of 0.1 to 5000 ng/kg bodyweight, particularly in an amount of 2 to 1000 ng/kg body weight andmore particularly in an amount of 3 to 600 ng/kg body weight of thesubject to be treated, depending on the type and severity of thecondition to be treated. In other embodiments of the present inventionthe soluble isoforms may also be administered locally, e.g. by directadministration into the central nervous system, e.g. into the spinalchord and/or into the brain. Also administration at higher dosages of upto 500 μg/kg by i.p. or s.c. injections, or inhalation devices are maybe considered. Preferably the subject to be treated is a mammal, morepreferably a human patient.

The soluble recombinant Neuregulin-1 isoforms may be administerd as astand-alone medication, i.e. as a monotherapy or as a co-medication,i.e. in combination with a further medicament, particularly with afurther medicament which is suitable tor the treatment of a neurologicalcondition. Examples of further medicaments are compounds affectingcatecholamine metabolism, acetylcholine esterase inhibitors, MAO-B- orCOMT-inhibitors, Memantine-type channel blockers, dopamine or serotoninereceptor agonists or antogonists, catecholamine or serotonine reuptakeinhibitors or any type of antipsychotic medicaments like clozapine orolanzapine or gabapentin-like drugs, particularly in the treatment ofAlzheimer's and Parkinson's diseases, schizophrenia, bipolar disorder,depression or other neurological conditions. Additional examples offurther medicaments are neuroprotective agents such as PARP-1inhibitors, e.g. as disclosed in WO 2006/008118 and WO 2006/008119,which are herein incorporated by reference.

Thus, an embodiment of the present invention refers to the combinationof a recombinant soluble Neuregulin-1 isoform as described herein with amedicament for the treatment of psychotic disorders such asschizophrenia, bipolar disorders and depression, e.g. olanzapine orclozapine. A further embodiment refers to the combination of arecombinant soluble Neuregulin-1 isoform and a medicament for thetreatment of a neurodegenerative disease such as Parkinson's disease,Alzheimer's disease, MS or ALS. Still a further embodiment refers to thecombination of a recombinant soluble Neuregulin-1 isoform and amedicament for the treatment of neurological injury, such as stroke,traumatic brain injury or spinal chord injury.

The combination therapy may be effected by co-administering therecombinant soluble Neuregulin-1 isoform and the further medicament inthe form of a pharmaceutical composition or kit, wherein the individualmedicaments are administered by separate or common administration.

The Neuregulin-1 isoform may be a Neuregulin-1 Type I, Type II, TypeIII, Type IV, Type V or Type VI isoform, preferably a Neuregulin-1 βisoform, a Neuregulin-1 α isoform or a Sensory and motor neuron-derivedfactor (SMDF) isoform, particularly a Neuregulin-1 β isoform and moreparticularly a human Neuregulin-1 β isoform.

Neuregulin 1 β isoforms are actively transported through the blood brainbarrier. The excellent bioavailability of Neuregulin 1 β in the brainafter i.v./i.p. Injection, as shown in the Examples paves the waytowards a therapeutic application of NRG 1 β.

Its combination of antiapoptotic, myelin-stabilizing, anti-inflammatoryproperties, together with the direct interaction with BACE opensopportunities in the treatment of stroke, Alzheimer, MS andschizophrenia and other neurological conditions.

As outlined above, the present application encompasses the use ofunmodified and modified Neuregulin-1 isoforms, particularly Neuregulin-1β isoforms. There is evidence that posttranslational modifications likeproteolytic processing, phosphorylation and glycosylation take place atcertain amino acid residues of the Neuregulin-1, and in particular itsextracellular domain. In particular the release of soluble fragments ofNeuregulin-1 has been reported (Buonanno and Fischbach 2001;Fischbach2007). Potential oxidation has been reported as well (Nadri et al.2007).

The present inventors have obtained evidence that preferredphysiologically active Neuregulin-1 β isoforms comprise theextracellular domain of Neuregulin-1 β or a part thereof which has beenpost-translationally modified. Preferably, the isoforms have beenmodified by phosphorylation, wherein 1, 2, 3 or more amino acid sidechain residues, particularly side chain residues having an OH-group suchas Tyr, Ser or Thr, have been phosphorylated. Preferred phosphorylationsites are located at amino acid positions 79-82, 133-136 and/or 158-161(nomenclature according to Falquet et al., 2002). Further preferredphosphorylation sites are located at amino acids 12-14, 30-32 and/or85-87. Further potential modification sites are amidation sites,preferably located at positions 22-25 and/or 30-33, glycosylation sitesat positions 150-153, 156-159 and/or 204-207, and myristylation sites,preferably located at positions 94-99, 149-154, 168-173, 175-180 and/or202-207 according to the nomenclature of Falquet et al. 2002.

In the following, the relevance of the experimental data according tothe present application are explained with regard to preferred medicalindications.

Schizophrenia

Schizophrenia is a serious and disabling mental disorder with symptomssuch as auditory hallucinations, disordered thinking and delusions,avolition, anhedonia, blunted affect and apathy. Epidemiological,clinical, neuropsychological, and neurophysiological studies haveprovided substantial evidence that abnormalities in brain developmentand ongoing neuroplasticity play important roles in the pathogenesis ofthe disorder (Arnold et al. 2005).

Schizophrenia is thought to include a disorder of dopaminergicneurotransmission, but modulation of the dopaminergic system byglutamatergic neurotransmission seems to play a key role. This view issupported by genetic findings of the neuregulin- and dysbindin genes,which have functional impact on the glutamatergic system (Muller andSchwarz 2006). What has become increasingly clear is that severalregions that are likely to contain genes (including neuregulins)contributing to schizophrenia are also relevant to bipolar affectivedisorder, a finding supported by recent twin data (Farmer et al. 2007;Owen et al. 2007).

Neuregulin-1, which is a psychosis susceptibility gene with effects onneuronal migration, axon guidance and myelination that could potentiallyexplain findings of abnormal anatomical and functional connectivity inschizophrenia and bipolar disorder (McIntosh et al. 2007).

There is an ever increasing body of evidence of a genetic linkage ofNeuregulin 1 to schizophrenia (review: Farmer et al., 2007). Theenhancement of glutamate, GABA and nicotinic neurotransmission byNeuregulin-1 (Fischbach 2007;Woo et al. 2007;Li et al. 2007) is relevantin this context, as well as implication with brain inflammation(Hanninen et al. 2007).

The regulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, therate-limiting enzyme for cholesterol biosynthesis (Pertusa et al. 2007),important for myelinisation, is assumed to have implications in thiscondition as well.

The fact that among genetic risk factors common to schizophrenia,bipolar disorder and depression, NRG1 plays an outstanding role, hastriggered suggestions that genes implicated in these psychoses such asNRG-1 may eventually provide the basis for classification based onbiology rather than symptoms, and lead to novel treatment strategies forthese complex brain disorders (Blackwood et al. 2007;Bertram et al.2007).

The experimental data of the present application demonstrate theeffectiveness of administration of a soluble recombinant Neuregulin-1 βisoform in an experimental model of schizophrenia.

Alzheimer's disease

Initial research by the inventors showed that Neuregulin 1 β isdiminished in post mortem sections of hippocampi of brains ofAlzheimer's patients as compared to age-matched controls (Sommer et al.,2004) with a clear positive correlation of the soluble fragment ofNeuregulin-1 with learning performance in a radial maze test (Sommer etal., 2004).

There are numerous reports demonstrating the role of NRG-1 inactivity-dependent synaptic changes (Xie et al. 2006;Kwon et al.2005;Rimer et al. 2005;Bao et al. 2004;Yang et al. 2005) important forlearning and memory (Ozaki et al. 1997;Ozaki et al. 2004;Golub et al.2004;Schillo et al. 2005b). As shown below, the NRG1β fragmentcontaining the extracellular domain was clearly associated with learningin a behavioural animal model. Showing decreased expression of theprotein in post mortem brain slices of the hippocampal regions(responsible for short term memory formation) of Alzheimer patients ascompared to age-matched controls could demonstrate the absence ofmemory-related synaptic activity, in regions of apparently still healthyneurons.

Very recent discoveries (Hu et al. 2006;Glabe 2006;Schubert 2006) showthat NRG 1 is processed by BACE1 (=β secretase), an enzyme that helpsgenerate clumps of amyloid-β in the brains of people with Alzheimerdisease, which explains the link to Alzheimer's disease, its concomitantrole in myelin formation relates to the neurotrophic properties of NRG 1(Hu et al., 2006; Glabe 2006; Schubert 2006). The enzyme, BACE1(beta-site amyloid precursor protein-leaving enzyme 1), is required tocleave amyloid-β from a larger precursor. (After BACE1-mediatedcleavage, the presenilin-containing complex y-secretase makes the finalcleavage, liberating amyloid-β.

The cleavage of NRG by secretases is crucial for nerve myelination. Justlike amyloid precursor protein, neuregulin 1 is also cleaved byβ-secretase. Proteolytic cleavage of neuregulin 1 by β-secretase iscritical for peripheral nerve myelination by Schwann cells. Drugs thattarget β-secretase could affect peripheral nerve development andfunction.

The in initial observation was by the group of Haass (Willem et al.2006), who found that BACE1 seems also to be required for myelination.Peripheral nerve myelination occurs early in life, so it is unclear howBACE1 inhibition might affect older animals. There are indications thatBACE1 also has a role in myelination of the central nervous system.Transgenic animals deficient in BACE-1 had myelin defects in theperipheral nerves

Also in the context of neurodegeneration and Alzheimer's disease, therecent discovery of enhancement of glutamate, GABA and nicotinicneurotransmission by Neuregulin-1 (Fischbach 2007;Woo et al. 2007;Li etal. 2007) is relevant.

The experimental data of the present application demonstrate theeffectiveness of administration of a soluble recombinant Neuregulin-1 βisoform in an experimental model of Alzheimer's disease.

Stroke, Traumatic Brain Injury

A series of stroke-related in vivo experiments by independent externalresearch in the US, demonstrate neuroprotection by Neuregulin 1 which byitself is antiapoptotic (Xu et al., 2004, 2005 and 2006; Guo et al.,2006)

NRG-1 reduces neuronal damage and improves neurological outcome aftermiddle cerebral artery occlusion (a common stroke model) (Xu et al.2005b;Xu et al. 2004;Xu et al. 2006;Guo et al. 2006).

In the same study about the therapeutic efficacy and mechanism ofrecombinant human NRG-1 in attenuating brain injury byischemia/reperfusion, it was found that NRG is antiapoptotic. NRG-1 (3.0ng/kg) was applied intravascularly 10 min before middle cerebral arteryocclusion (MCAO) and subsequent focal cerebral ischemia for 90 min andreperfusion for 24 h.

The data of the present invention demonstrate that administration ofrecombinant soluble Neuregulin-1 isoforms at low concentration has asignificant pharmacological effect and thus is assumed to be effectivein models of stroke and traumatic brain injury.

In the following, the present application is explained in more detail bythe Figures and Examples given herein below.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: Various reviews and numerous research articles on Neuregulin 1show the key functional position of NRG 1 as an upstream regulatoryprinciple of mechanisms thought to be pivotal in neurodegenerativediseases, neurological disorders, as well as physiological function:

NRG are key parts of functional complexes, consisting at least ofneuregulins (NRG), receptor tyrosine kinases (ErbB receptors),heparansulfate proteoglycans (HSPG) and NMDA receptors (NMDAR), whichare transiently and activity-dependent assembled together in cholesterol(CHO)-rich membrane microdomains. In particular the shaping of calciumsignals is important for the interaction with subsynaptic scaffoldingproteins by posttranslational modifications (PSD-95, by interaction withcertain phosphorylated domains, like PDZ- or SH-domains on partnerproteins). The PSD-95 complex directly regulates pro-inflammatoryenzymes like nitric oxide synthase (NOS, iNOS is inducible, nNOS isneuronal) and Cox-2 (cyclooxygenase-2), which promote their effects in acomplex relationship with related, but not necessarily downstreammechanisms, involving NAD⁺-dependent enzymes like PARP-1(poly-ADP-ribose polymerase-) and Sir-2 (sirtuin-2); PARG ispoly(ADP-ribose) glycohydrolase the complementary and antagonisticenzyme to PARP-1, HDAC are histone deacetylases, the general class ofenzymes which includes Sir-2. MPTP stands for the mitochondrialpermeability transition pore. DRP-2 is dihydropyrimidinase-relatedprotein 2. Also other important membrane proteins, like certainnicotinic acetylcholine receptors (nAChRα7), GABAA receptors (GABA_(A)R)amyloid precursor protein (APP) and proteases (PS) are transientlyorganized in lipid rafts and acquire different functional propertiesoutside the usual phospholipid (PL) environment, details in(Schrattenholz and Soskic 2006).

FIG. 2: Summary of learning experiments in Morris water maze: animalstreated with a daily dose of 3 ng/kg (i.v.) of the soluble extracellulardomain of neuregulins-1 beta (NRG-1 beta-ECD) were significantly betterin learning than vehicle treated animals; IAE: inner area entry; IAEFinner area entry frequency; TS: time spent in inner area; DT: distancetravelled in inner area.

FIG. 3: Reduction of Amphetamine-induced hyperactivity by NRG-1beta-ECD, a widely accepted model for schizophrenia. Concentrationsranged from 15 to 600 ng/kg (i.v. injection 15 minutes prior toamphetamine application). A positive control of 0.125 mg/kg Haloperidolwas included. Whereas Haloperidol like other non-typical and typicalantipsychotics usually reduce activity below control level (indicatedhere by dotted lines labelled veh/veh, in blue for crossings and inmagenta for rears), NRG-1 beta-ECD reduction asymptotically approachescontrol levels of activity, but does not cause further reduction. Thelow effective concentrations of NRG-1 beta-ECD and the absence ofnegative effects (reduction of activity below vehicle control levels)are the outstanding properties in this model. The effects aresignificant with p<0.05;

FIG. 4: Summary of learning experiments with APPPS mouse model ofcerebral amyloidosis and Alzheimer's disease in a Morris water maze:animals treated with a daily dose of 200 ng/kg i.p. NRG-1 beta-ECD weresignificantly better in learning than vehicle treated animals; IAE:inner area entry; IAEF inner area entry frequency; TS: time spent ininner area; DT: distance travelled in inner area.

FIG. 5: HPLC quantification of dopamine and its metabolites: The columnslabelled with asterisks are highly significant.

Legend S Saline (control) aM acute MPTP aMN acute MPTP and NRG-1beta-ECD aN acute NRG-1 beta-ECD cM chronic MPTP cMN chronic MPTP andNRG-1 beta-ECD cN chronic NRG-1 beta-ECD

FIG. 6 Metabolism of dopamine by MAO-B and COMT.

FIG. 7 MPTP exposition leads to a significant loss of dopaminergicneurons in the substantia nigra (aMPTP, p=0.0005; and cMPTP, p=0.0075).The ip application of 20 ng/kg of NRG-1 beta-ECD leads to a reversal(aNR-MPTP; p=0.57, i.e. not different from vehicle control) or clear andsignificant improvement of the MPTP lesion (cNR-MPTP; p=0.0097); in thechronic model (5 days daily ip application of 20 ng/kg of NRG-1beta-ECD) there is also a significant effect number of dopaminergicneurons (cNR; p=0.0002);

Legend NaCl Saline (control) aMPTP acute MPTP aNR-MPTP acute MPTP andNRG-1 beta-ECD aNR acute NRG-1 beta-ECD cMPTP chronic MPTP cNR-MPTPchronic MPTP and NRG-1 beta-ECD cNR chronic NRG-1 beta-ECD

FIG. 8 Two representative images of 2D-Western blots of brain proteinsof APPPS mice stained for Neuregulin-1β are shown of each, a treated andgood learning animal (top) and non-treated animal with inferior learningperformance (below).

The numbers in the upper part are pl values of the 2D gel.

FIG. 9 A Western blot experiment compairs the abundance of the NRG-1βECD-fragment in post mortem cortical material from Alzheimer patientsand controls.

FIG. 10 2D-PAGE shows, that the acidic isoform of NRG-1 β-ECD, with a pIof approx. 5-5.5 and a molecular weight of approx 25-32 kD in theseexperiments is clearly diminished in Alzheimer's patients brains.

FIG. 11 is a schematic of the pool used for the Morris Water Mazeassessment.

FIG. 12 is a schematic of the pool used for the Morris Water Mazeassessment showing the zones for computing the animals' track records.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES General:

In all of the following experiments fragments of Neuregulin-1 beta havebeen used, comprising only the extracellular domain (ECD) of the entiretranscript of the human nrg-1 gene. They had a molecular weight ofapprox 25-32 kD and isoelectric points between approx 5 and 9.5,depending upon phosphorylation and/or glycosylation status.

The physiologically active form of Neuregulin-1 isoform has a pI ofapprox.5.5. The physiologically active form has an pI of approx.5.5(most of the experiments were carried through with a commerciallyavailable isoform produced in E. coli, with a molecular weight of 26 kDand an pI of approx.9.0)

This isoform is a recombinant soluble human NRG-1 beta fragmentconsisting of the first 245 amino acids of NRG-1B, purchased from R & DSystems, Inc. (Catalog No. 377-HB-CF). It will be named NRG-1 beta-ECDin the following. This active isoform has a pI of approx. 9.0

We also tested a corresponding fragment of NRG-1β with 8 kD, onlycomprising the EGF domain, purchased from R&D Systems (Catalog No.396-HB). This fragment appears to be neuroprotective as well in vitroand in vivo, but was not investigated in depth because of much higherproliferative properties, which raised concerns about cancerogenity.

Example 1

Initial Toxicology Data Indicate that NRG1β (ECD) has no Adverse Effectsin Acute Toxicology and In Vitro Mutagenicity Tests.

-   -   There was no acute intravenous toxicity in rats: All animals        survived until the end of the study period. No clinical signs        were observed during the course of the study. The body weight of        the animals was within the range commonly recorded for this        strain and age. No macroscopic findings were recorded at        necropsy. The median lethal dose of NRG1β (ECD) after single        intravenous administration to female rats, observed over a        period of 14 days is: LD₅₀ (female rat): greater than 5000 ng/kg        body weight.    -   Daily intravenous administration of Neuregulin over a period of        seven days at dose levels of 50, 200 and 600 ng/kg body weight/d        did not result in any premature death. No clinical signs were        recorded. The treatment did not affect the food consumption and        body weight development. The no observed effect level (NOEL) was        established at 600 ng/kg body weight/d.    -   In the mouse lymphoma thymidine kinase locus assay using the        cell line L5178Y according to the OECD Guideline for the Testing        of Chemicals, No. 476 “In vitro Mammalian Cell Gene Mutation        Test”, NRG1β (ECD) was non-mutagenic.    -   In the chromosome aberration test in Chinese hamster V79 cells        according to the OECD Guideline for the Testing of Chemicals,        No. 473, NRG1β (ECD) did not induce structural chromosome        aberrations.

Moreover, in none of the animal experiments carried out with regard toefficacy (some of them going on for several months with daily ivapplications) did we ever observe adverse effects of NRG1β (ECD).

Application of NRG1β (ECD) in the various animal models described below,was either by intravenous (iv) or intraperitoneal (ip) injections;concentrations were ranging from 3-600 ng/kg.

Example 2

Learning and Mmemory: Spatial Learning with and without NRG-1 beta-ECDApplication

Methods:

The Morris Water Maze assesses spatial learning. It requires animals toswim in a water-filled pool and to find a rescue platform submerged justbelow the surface. It is obligatory that the platform is placed awayfrom the walls of the maze and that animals have reference pointsvisible from the water surface that permit estimation of location, butare not close enough to the target to permit associative learning. Theanimals are trained that rescue only comes via the platform meaning thatall animals which do not find the platform, are guided to the platformand allowed to rest before being removed from the set-up. Therefore, oneof the most important reference points for the mouse is the humanoperator.

The experiment aims at determining two key parameters associated withmurine spatial recall:

-   -   the rate at which the mice learn to relocate the platform    -   the ability to retain the information in the short term (within        a training period or overnight)

Animals

The study is performed with two groups of APP/PS mice (Meyer-Luehmann etal. 2006;Radde et al. 2006), one of which is treated by a daily dose ofNRG-1 beta-ECD and the other one is sham treated as a control. Eachgroup consists of 8 males which are nine weeks old at the beginning oftheir first series of experiments.

The first series of experiments started with two subgroups of 8 treatedand 8 untreated mice on week 42 and will last for 15 days. Furtherseries of identical experiments will be performed 6, 12, etc. weekslater.

For a second pair of subgroups (8 treated and 8 untreated nine weeks oldmales) the same series of experiments started on week 48, so theexperiments of these subgroups lag exactly 6 weeks behind the ones ofthe first subgroups.

Apparatus

The learning aptitude of the treated and untreated APP/PS mice isassessed using a circular Morris water maze which should be large enoughto provide searching space without exhausting the mouse. Utmost careneeds to be taken to keep each detail of the experimental setup asinvariable as possible throughout all experiments.

In the current study, a pool of 120 cm diameter is used which is placedat an exactly reproducible position in the lab with always identicalorientation. At fixed positions in the pool, a white, translucent,circular platform of 15, 10, or 5 cm diameter is placed that extends tojust below the water surface (so it is invisible to the mice) and thatthe animals can climb on—which is the only means to rest out of thewater. To assist climbing, the platform is coated with a gauze gripsurface (see FIG. 11).

In order to perform the rescue procedure in probe trials as detailedbelow, the platform is equipped with a mechanism that allows forautomatically raising and lowering it without direct operatorintervention. Thus, depending on its height the platform is accessibleto the swimming mice or not “On-demand platform” (Buresova et al. 1985).

Platform locations are always situated in a ring shaped, concentricregion of the pool with inner and outer diameters ˜40 cm and ˜80 cm,respectively. Four quadrants are defined such that the platform occupiesthe central region of one of them (the target quadrant). For furtherdetails on platform sizes and positions see below.

In order to make sure that platform position is exactly the samethroughout an entire series of experiments; a socket will be firmlyaffixed to the floor of the pool on which the platform can be mountedwith a minimum of spatial tolerance. On top of the platform, in itscenter, there is another mounting for a (proximal) cue sticking out ofthe water which is well visible on the video recording as well as to themice swimming in the pool. For a check of platform position, a briefvideo recording will be taken without an animal but with the cue pluggedinto the platform whenever the platform or the video camera have beenmanipulated with in any way.

The water is made opaque using low fat milk powder. The watertemperature should be cold enough to encourage searching for an exit butnot so cold that the animals suffer or are exhausted. As a faircompromise, water temperature is monitored at the start of eachexperiment and modulated with either warm water or ice flakes to 18° C.Between individual trials, temperature is readjusted as needed.

Four distal cues (of different simple geometric shapes and differentcolors, height ˜20cm) are attached ˜20cm above the sides of the pool,one in each quadrant. Care is taken to place each cue in exactly thesame location throughout all experiments. The entire pool is enclosed bya white translucent curtain. Lighting is dimmed and diffuse.

A video camera is firmly mounted at an exactly vertical position abovethe center of the pool, such that the pool completely fills the videoimage. Video recordings are taken at PAL resolution (720×576 pixels, 25frames per second), at the least. The videos are evaluated by anautomatic tracking system that allows for flawless detection of theanimals movements with time.

Mice are placed into the water using a special devise that is mounted ona stick, so they can be watered at exactly defined spots along the rimof the pool without the operator entering the cabin made up of thetranslucent curtain.

Experimental Design

In each session, mice are placed into the pool at predefined sites andare allowed to swim for 60s. Animals' motion tracks are recorded by avideo tracking system, and parameters are computed from whichconclusions regarding the animals' learning aptitude can be drawn (mostnotably the period until the mouse hits the platform for the firsttime=“escape latency”; further details see below). If a mouse succeedsin finding the platform it is left resting there for a short period oftime (˜15 s). Otherwise, after 60s of swimming, the mouse is guided tothe platform by the operator and allowed to rest for ˜15s. Afterwards,it is picked up by the operator, dried gently and returned to itshousing or prepared for the next swim.

On each day of experiments, one trial per mouse is performed in theearly morning. Each trial consists of two consecutive swims originatingfrom two different quadrants, but never from the target quadrant. Exactwatering sites (and platform positions whenever applicable) are assignedrandomly for each swim of each day, but do not differ between theindividual mice during that day.

If mice turn out to learn extremely slowly the number of swims per trialor trials per day may be increased (and vice versa). Moreover, in manymouse strains younger animals learn very quickly, so after four or fivedays of training, escape latencies remain constant at a few seconds onlywhich is equally true in treated as in untreated animals. However, forthe statistical evaluation, it is of advantage if the curve of escapelatencies over training days does not saturate but rather decreasesmonotonically. Therefore, an experimental design is used in which theproblem to solve becomes more difficult with training progress: Onpredefined days the platform is replaced with a smaller one while theplatform's center coordinates remain the same. If and when platforms areswapped may be determined independently for each series of experimentsand ought to depend on the outcomes of the preceding series.

In each series of experiments mice are subjected to three differentkinds of tasks:

-   -   Cued place navigation. The platform is marked with a cue, and        the mouse is allowed to swim until it finds the platform. This        procedure tests associative learning and serves for dividing        mice into two experimental groups the learning aptitudes of        which are as similar as possible. Moreover, in the second and        further series of experiments, cued place navigation supports        blanking recollection of the position of the platform in        preceding series.    -   Hidden platform acquisition training. The platform is invisible        to the mouse and located at the same position as during the        preceding swim. This task allows for monitoring the mouse's        progress in recalling the exact location of a hidden platform        (“spatial learning”).    -   Probe trial testing. In this task, the on-demand platform is        maximally lowered underneath the surface and the mouse is        allowed to swim freely searching for it. Probe trial testing        assesses the animals' absolute recall which, in this context,        can be also interpreted as conviction, persistence or certainty        regarding the platform location. The conventional approach to        interpreting the experiment is that animals that have firmly        fixed the location of the platform will more persistently search        in a limited location and thus spend more time in the zone next        to the platform.    -   In probe trial testing, there is a risk that the inability to        find the plat-form may reduce the incentive to swim to the        platform zone. In order to keep these irritations as small as        possible, the modalities of human rescue ought to remain the        same so there is some spatial constancy despite the absence of        the platform. Therefore, after 60s of swimming the platform is        lifted to just beneath the surface, the mouse is guided there by        the operator and allowed to rest for ˜15s before being taken out        of the apparatus.    -   On all days of probe trial testing, only one swim is performed.

Approximately 60 minutes prior to each trial, mice are treated dailywith either 5 ng/kg NRG-1 beta-ECD (suspended in black 6 mouse serum andprovided i.v. in a volume of 20 82 L per mouse) or with 20 82 L ofvehicle i.v., respectively.

On day 1 of the first series of experiments, all mice in the studyreceive sham treatment only. Thereafter, mice are assigned to theNeuregulin and the control groups such that the distributions of escapelatencies match in both groups.

In each series of experiments the following chronology is adhered to:

-   -   day 1. Cued platform search with platform of size 10 cm and        position changing for each swim.    -   day 2. Cued platform search with platform of size 10 cm and        position changing for each swim.    -   day 3. Cued platform search with platform of size 10 cm and        position changing for each swim.    -   day 4. Cued platform search with platform of size 10 cm and same        position as the last one on day 3.    -   day 5. Hidden platform search with platform of size 15 cm and        same position.    -   day 6. Hidden platform search with platform of size 15 cm and        same position.    -   day 7. Hidden platform search with platform of size 15 cm and        same position.    -   day 8. Hidden platform search with platform of size 10 cm and        same position.    -   day 9. Probe trial testing.    -   day 10. Hidden platform search with platform of size 10 cm and        same position.    -   day 11. Hidden platform search with platform of size 10 cm and        same position.    -   day 12. Hidden platform search with platform of size 5 cm and        same position.    -   day 13. Hidden platform search with platform of size 5 cm and        same position.    -   day 14. Hidden platform search with platform of size 5 cm and        same position.    -   day 15. Probe trial testing.

It may be necessary to aid unlearning of the platform position from apreceding set of experiments by allowing the mice to freely swim for afew days without platform present.

The rate of learning is assessed by monitoring each training/testsession and noting the success of the animals in finding the platform aswell as the evolution of the search strategy from skirting the sides ofthe pool to moving away from the sides to search in the near to centralarea where the platform lies.

Measured Parameters

From the animals' video recordings, each mouse's motion track isextracted and exported as a series of x, y, and time coordinates forfurther processing. Care needs to be taken to reliably identify eachtrack's staring point and to avoid tracking errors. Simultaneously, anumber of parameters are computed from which conclusions regarding theanimals' learning aptitude can be drawn (see below). Parameterrecordings are halted after 60s or if the mouse has found the platform(whichever happens earlier).

For the definition of parameters to be computed from the animals' trackrecords the following zones are defined (see FIG. 12):

In order to keep evaluations as flexible as possible, four concentrictarget zones (centered about the platform) of 5.5 to 30 cm diameter areemployed. Parameters computed from the animals' track records include:

-   -   Total distance travelled    -   Overall average speed    -   Number of entries to the pool center    -   Time in the pool center    -   Latency to first entry to the pool center    -   Distance travelled to first entry to the pool center    -   Number of entries to the inner area    -   Time in the inner area    -   Distance travelled in the inner area    -   Latency to first entry to the inner area    -   Distance travelled to first entry to the inner area and for each        target zone 1 to 4 and the target quadrant    -   Number of entries to the zone    -   Time in the zone    -   Distance travelled in the zone    -   Latency to first entry to the zone    -   Distance travelled to first entry to the zone    -   Distance from beginning of track to nearest point of zone    -   Average distance from the zone when outside the zone    -   Minimum distance from the zone when outside the zone    -   Time to minimum distance from the zone when outside the zone    -   Time getting closer to the zone    -   Time getting further away from the zone    -   Time moving towards the zone    -   Time moving away from the zone    -   Number of head entries to the zone    -   Time of head in the zone    -   Distance of head travelled in the zone    -   Latency to first entry of head to the zone    -   Average distance of head from the zone when outside the zone    -   Minimum distance of head from the zone when outside the zone    -   Initial heading error    -   Average heading error    -   Number of exits from the zone

For each day of the experiment, the readings of parameters of learningprogress in the treated and untreated groups are compared to each otherstatistically.

On examining the track records of individual mice, a human observer isable to come to a fairly realistic perception of the animals'assertiveness in locating the platform which is not fully reflected inthe measured parameter values. Therefore, track records are alsomanually inspected and the animals' recall of the platform position israted.

Results:

Those animals treated with a daily dose of 3 ng/kg NRG-1 beta-ECD i.v.30 min prior to training were significantly better in learning-relatedparameters than the vehicle treated group.

Neuregulin not only improved learning, but treated animals had alsodeveloped more advanced search strategies: More treated animals enteredthe inner area of the pool (11 vs. 7, p=0.019), entries to the innerarea occurred more often (2.17 vs. 0.92 times, p=0.02), time spent anddistance travelled in the inner zone was longer (6.51s vs. 2.13s, p=0.09and 0.64m vs. 0.25 m, p=0.031, respectively).

The results of the learning experiments in a Morris water maze aresummarized in FIG. 2.

Example 3 Schizophrenia: Amphetamine-Induced Hhyperactivity in the Rat

Methods: The method, which detects antipsychotic and anti-Parkinsonactivity, follows that described by Costall et al. 1978 and uses anactivity meter similar to that described by Boissier and Simon 1966.

Amphetamine induces hyperactivity in this test situation. Hyperactivityis antagonized by classical and atypical antipsychotics acting ondopaminergic systems at the limbic level, and is potentiated byanti-Parkinson drugs.

Rats are injected with d-amphetamine (3 mg/kg i.p.) and are immediatelyplaced in the activity meter.

The activity meter consists of 12 covered Plexiglass cages (40×25×25 cm)contained within a darkened cabinet. Each cage is equipped with twophotocell assemblies at each end of the cage, 3 cm above the floor, inorder to measure the number of movements by each animal (one per cage)from one end of the cage to the other. Two additional photocellassemblies are placed at 20 cm above the floor to record rearing. Thescores for activity and rearing are recorded by computer over 10-minuteintervals and cumulated over a 30-minute period.

15 rats were studied per group. The test was performed blind.

The test substance was evaluated at 8 doses, administered i.v. 15minutes before amphetamine, and compared with a vehicle control group.The experiment also included a control group not treated withamphetamine.

Haloperidol (0.125 mg/kg i.v.), administered under the same experimentalconditions, was used as reference substance.

The experiment therefore included 16 groups.

Data were analyzed by comparing treated groups with appropriate controlusing unpaired Student's t tests.

Results:

As shown in FIG. 3, NRG-1 beta-ECD in a dose-dependent manner inhibitsthe amphetamine-induced hyperactivity in an animal model forschizophrenia. Conspicuously, the experiments reveal outstandingproperties of NRG-1 beta-ECD:

-   -   The effects shown in FIG. 3 are strongest in the second half of        the experiment (minutes 20-40). In the first 20 minutes only a        smaller effect can be found, this delayed effect points to        further processing of the protein.    -   The effective concentrations of NRG-1 beta-ECD used here are        about 200-1000 times lower than those used for typical control        neuroleptica like Haloperidol (125 μg/kg).    -   In contrast to Haloperidol, Clozapine, Olanzapine etc. there are        no negative effects observed in that NRG-1 beta-ECD does not        reduce activity of test animals below vehicle control levels.

Example 4 Schizophrenia: Prepulse Inhibition

Rodents with NRG1 knock-out show significantly impaired prepulseinhibition (PPI) linking NRG1 to schizophrenia. A widely used surrogatemeasure of psychosis in animal models, PPI is considered a schizophreniaendophenotype. It was reported that there are neurophysiological effectsof missense mutations of a nonsynonymous single nucleotide polymorphismlocated on NRG1 (rs3924999) on PPI after extensive genotyping, in bothschizophrenia and healthy control populations (Hong et al. 2007). Wetested the effect of NRG-1 beta-ECD on PPI. The results so far may besummarized as follows:

At 105 dB, NRG-1 beta-ECD showed a general trend towardsre-establishment of PPI (+26%, +23% and +36%, at 150, 300 and 600 ng/kgrespectively), although the effect did not reach statisticalsignificance and was not observed at 115 dB. It had no effects onspontaneous movements in the absence of stimulus at 150 or 300 ng/kg butsignificantly decreased spontaneous movements in the absence of stimulusat 600 ng/kg (−20% and −29%, on average and peak intensitiesrespectively, p<0.05, this is similar to aripiprazole). NRG-1 beta-ECDhad no effects on the reaction to the pre-pulse alone.

The results so far suggest the absence of significant effects onapomorphine-induced PPI deficits for Propsyl00 over the dose-range150-300 ng/kg and a decrease of spontaneous movements as well as a trendtowards re-establishment of PPI at 600 ng/kg i.v. in the Pre-pulseInhibition (PPI) Test in the rat (deficits induced by apomorphine).

In this series of experiments, the reference substance, aripiprazole,had weak but significant activity at 3 mg/kg i.p., but not at 10 mg/kgi.p., in the same test.

All together and under conditions used, NRG-1 beta-ECD appears to affectPPI at higher concentrations around 600 ng/kg. These resultssurprisingly open a novel understanding of recent neurobiologicalresearch implying (NRG1) as one of the leading candidate genes inschizophrenia.

Example 5 Learning and Memory in an Animal Model for Alzheimer's Disease(APPPS dt Mice)

The animal experiments testing learning and memory with or withoutapplication of the soluble extracellular domain of Neuregulin 1 β (NRG-1beta-ECD) in a Morris water maze set up described above for normal mice,have been repeated double transgenic mouse model for cerebralamyloidosis (APPPS mice(Meyer-Luehmann et al. 2006;Radde et al. 2006)).

Here again those animals which were treated with a daily dose of NRG-1beta-ECD (here 200 ng/kg i.p. were applied) 30 min prior to trainingwere significantly better in learning-related parameters than thevehicle treated group.

Neuregulin not only improved learning, but treated animals had alsodeveloped more advanced search strategies: More treated animals enteredthe inner area of the pool (12 vs. 7, p=0.009), entries to the innerarea occurred more often (2.0 vs. 0.7 times, p=0.03), time spent anddistance travelled in the inner zone was longer (5.3 s vs. 2.1 s, p=0.09and 0.7m vs. 0.3 m, p=0.025, respectively).

The results of the learning experiments with APPPS mouse model ofceretral amyloidosis and Alzheimer's disease in a Morris water maze aresummarized in FIG. 4.

Example 6 Neuregulin 1-Beta MPTP Mouse Model of Parkinson's DiseaseMethods:

Male C57B1/6 mice of 10 weeks were used in the MPTP(1-Methyl-4-phenly-1, 2, 3, 6-tetrahydropyridine) model for Parkinson'sdisease.

Brain tissue is dissected (Substantia Nigra, Striatum, Cortex) of 10weeks old male C57B16 mice (N=10 per group) at different times aftertreatment (0, 1, 3, 7, 21 days) with NaCl (controls) or MPTP (acute andsubchronic models). The methods follow published procedures (Hoglingeret al. 2007;Hoglinger et al. 2004).

Time after injection of MPTP Treatment 0 days 1 days 3 days 7days 21days MPTP acute N = 10 N = 10 N = 10 N = 10 N = 10 MPTP chronic N = 10 N= 10 N = 10 N = 10 N = 10 NaCl N = 10 Total: N = 110

MPTP is dissolved as a powder in 0.9% NaCl and is injectedintraperitoneal

(acute application: 4×20 mg/kg, each at 2 hour intervals; chronicapplication: 5×30 mg/kg, each at 24 hour intervals). These injectionstake approx. 10 seconds, animals were sacrificed at defined time points(see table) by cervical dislocation. The procedures follow publishedprotocols (Hoglinger et al. 2007;Hoglinger et al. 2004;Liberatore et al.1999;Przedborski and Vila 2003;Vila and Przedborski 2003).

0 days after last MPTP-administration: Loss of striatal dopaminergicnerves

1 day after last MPTP-administration: Beginning microglia-activation

3 days after last MPTP-administration: Maximum of microglia-activation

7 days after last MPTP-administration: Maximum of astrocyte-activation

21 days after last MPTP-Administration: Maximum of cell death

21 days after intracerebral infusion of NRG-1 beta-ECD and a controlpeptide via Alzet Mini pumps, followed by MPTP treatment (acute vs.chronic), a histological quantification of dopaminergic neurons of themiddle brain was performed according to stereological principles. Also,a biochemical quantification of dopamine and its metabolites in thestriatum is performed by HPLC. Procedures are performed according topublished protocols (Hoglinger er et al. 2007;Hoglinger et al. 2004).

Treatment Infusion N MPTP acute NRG-1 beta-ECD N = 10 Control acuteNRG-1 beta-ECD N = 10 MPTP chronic NRG-1 beta-ECD N = 10 Control chronicNRG-1 beta-ECD N = 10 MPTP acute Control peptide N = 10 Control acuteControl peptide N = 10 MPTP chronic Control peptide N = 10 Controlchronic Control peptide N = 10 Total: N = 80

Results:

As shown in FIG. 5, the results of the HPLC measurements of dopamine andits metabolites reveal a clear effect of administration of NRG-1beta-ECD during MPTP insult, in this model for Parkinson's disease.

The effects are non-classical: whereas there is no significant effectupon dopamine levels, neither during MPTP-insult nor in acute or chroniccontrols of NRG-1 beta-ECD administration, there are pronounced andclear effects on the concentrations of DOPAC and HVA. The chronicadministration of NRG-1 beta-ECD results in a clear and significantreduction of this metabolite in the absence of MPTP insult, whereas inthe acute regimen only a slight decrease is observed. During the chroniccondition of the MPTP insult, NRG-1 beta-ECD causes a significantincrease of homovanillic acid (HVA), an effect, which is even morepronounced in the absence of MPTP-insult.

These results are can be interpreted by a down regulation of MAO-Bduring the chronic NRG-1 beta-ECD administration and/or COMT upregulation. Under the conditions applied a huge and significant positiveeffect on survival of dopaminergic neurons was observed. NRG-1 beta-ECDis also highly neuroprotective in this model. Given the ip injectionduring this series of experiments, the clear efficacy also proves againthat NRG-1 beta-ECD is highly efficient in passing the blood brainbarrier.

FIG. 6 shows the metabolic scheme which appears to be affected by NRG-1beta-ECD administration: dopamine is converted by MAO-B to DOPAC and byCOMT to 3-MT; homovanillic acid is subsequently generated form bothmetabolites by COMT from DOPAC and by MAO-B from 3-MT;

NRG-1 beta-ECD administration is obviously regulating activities of bothenzymes.

Even more important and as shown in FIG. 7, in the MPTP model ofParkinson's disease there is a clear and significant neuroprotectiveeffect of NRG-1 beta-ECD becoming apparent by histologicalquantification of dopaminergic neurons of the middle brain. Thestereological method has been described elsewhere (Liberatore et al.,1999, Przedborski & Vila, 2003; Vila & Przedborski, 2003; Hoglinger etal., 2004; Hoglinger et al., 2007).

Taken together, there is a surprisingly clear and beneficialneuroprotective effect in the MPTP animal model of Parkinson's disease:The effects prove again that the intraperitoneal administration of verylow concentrations of NRG-1 beta-ECD (e.g. 20 ng/kg) is sufficient toachieve efficacy and thus that NRG-1 beta-ECD passes the blood brainbarrier. Also the complex influence on dopamine metabolites (HPLCresults; FIG. 5) points to regulation of MAO-B and COMT by NRG-1 andNRG-1 beta-ECD.

Example 7 Identification of an Acidic Posttranslational Iisoform ofNRG-1 Beta-ECD as the Active Principle

We have published evidence, that in learning and memory a particularposttranslational acidic isoform of NRG-1 beta-ECD is the active form(Schillo et al. 2005a). Here we show that similar patterns are observedin animal models of Alzheimer's disease and post-mortem brain tissuefrom Alzheimer's and Parkinson's disease patients. We conclude that thisacidic isoform is the active principle.

Methods:

For staining Western blots we used the following antibodies:anti-NRG1-ECD, rabbit polyclonal (sc-28916 Lot: I 2905 Santa Cruz;H-210)

Neuregulin-1 (H-210) is a rabbit polyclonal antibody raised againstamino acids 21-230 mapping within an N-terminal extracellular domain ofNeuregulin-1 isoform HRG-a of human origin. Neuregulin-1 (H-210) isrecommended for detection of Neuregulin-1 isoforms HRG-α, HRG-α1A,HRG-α2B, HRG-α3, HRG-β1, HRG-β2, HRG-β3 (GGF), GGF2 and SMDF of mouse,rat and human origin by Western Blotting (starting dilution 1:200,dilution range 1:100-1:1000), immunoprecipitation [1-2 μg per 100-500 μgof total protein (1 ml of cell lysate)] and immunofluorescence (startingdilution 1:50, dilution range 1:50-1:500).

Secondary antibodies were:

Anti goat, HRP

sc-2922 Lot: C1405 Santa Cruz

Anti rabbit, HRP

Sc-2054 Lot: G 2005 Santa Cruz

Next to immunostaining we performed MALDI-TOF and Q-TOF massspectrometry to confirm NRG-1 beta-ECD.

Now we find a very similar pattern in APPPS mouse model of cerebralamyloidogenesis and Alzheimer's disease as shown in FIG. 8. Theconcentration of this particular acidic isoform of NRG-1 beta-ECD atroughly a pI of 5.0 is considerably higher in treated APPPS mice whichare at the same time better learners.

In FIG. 8 two representative images are shown of each, a treated goodlearning animal (top) and non-treated animal with inferior learningperformance. (below).

FIG. 9 shows the results of a Western Blot experiment using post mortemcortical material from each 9 Alzheimer patients and age-matchedcontrols. It clearly reveals, that the NRG-1 β-ECD fragment issignificantly less abundant in the Alzheimer cases. As an internalcontrol the abundance of NRG-12 was measured, which appears not to beaffected by the memory-loss associated with the disease.

A further investigation of the this specific Alzheimer- andmemory-associated isoform of NRG-1 β-ECD by Western blots of2-dimensional gels (2D-PAGE) of the same post mortem human brainmaterial used for FIG. 9, reveals as shown by representative examples inFIG. 10, that it is indeed the acidic isoform of NRG-1 β-ECD which isdiminished in the Alzheimer condition.

Conclusions

We present here for the first time functional evidence of in vivoeffects of posttranslational modifications of the transcript of nrg-1gene, in particular a truncated form generated by proteolytic cleavage,comprising the extracellular domain of NRG-1 beta with MW 15-35, pI4-10; more specifically we found an antipsychotic activity in animalmodels for schizophrenia, probably based on regulation of MAO-B andCOMT, at concentrations of 5-600 ng/kg (i.v.). In contrast to controlneuroleptics which are used at concentrations which are 100-1000-foldhigher, there was no negative effect observed.

Moreover we found a neuroprotective effect in MPTP model of Parkinson'sdisease at concentrations of 3-300 ng/kg (i.v.)

Moreover we found a positive effect on memory- and learning inrespective animal models (Morris water maze) for learning and cerebralamyloidosis and Alzheimer's disease

Given the adverse effects of many atypical antipsychotics, currently inuse (Haddad and Sharma 2007) we conclude that soluble NRG-1-ECD fragmentwith EGF domains of SMDF, NRG-1 alpha, but in particular NRG-1 betamight be useful as a stand-alone or co- medication for the treatment ofschizophrenia, bipolar disorder and depression.

It might also be used in the same sense in other diseases of the centralnervous system, like neurodegenerative disorders like Alzheimer's andParkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,stroke, traumatic brain and spinal chord injuries.

Soluble NRG-1-ECD proteins have these very broad effects due to acentral role in neuronal signal transduction, in particular mediatingglutamate signalling and excitotoxicity, which plays a central role inall indications mentioned above (Schrattenholz and Soskic 2006).

REFERENCES

-   1. Arnold S E, Talbot K, Hahn C G (2005) Neurodevelopment,    neuroplasticity, and new genes for schizophrenia. Prog Brain Res    147:319-345-   2. Bao J, Lin H, Ouyang Y, Lei D, Osman A, Kim T W, Mei L, Dai P,    Ohlemiller K K, Ambron R T (2004) Activity-dependent transcription    regulation of PSD-95 by neuregulin-1 and Eos. Nat Neurosci    7:1250-1258-   3. Benzel I, Bansal A, Browning B L, Galwey N W, Maycox P R,    McGinnis R, Smart D, St C D, Yates P, Purvis I (2007) Interactions    among genes in the ErbB-Neuregulin signalling network are associated    with increased susceptibility to schizophrenia. Behav Brain Funct    3:31-   4. Bertram I, Bernstein H G, Lendeckel U, Bukowska A, Dobrowolny H,    Keilhoff G, Kanakis D, Mawrin C, Bielau H, Falkai P, Bogerts    β (2007) Immunohistochemical evidence for impaired neuregulin-1    signaling in the prefrontal cortex in schizophrenia and in unipolar    depression. Ann N Y Acad Sci 1096:147-156-   5. Blackwood D H, Pickard B J, Thomson P A, Evans K L, Porteous D J,    Muir W J (2007) Are some genetic risk factors common to    schizophrenia, bipolar disorder and depression? Evidence from DISC1,    GRIK4 and NRG1. Neurotox Res 11:73-83-   6. Boissier J R, Simon P (1966) [On the potentiation of DOPA effects    by monoamine oxidase inhibitors]. Psychopharmacologia 8:428-436-   7. Britsch S (2007) The neuregulin-I/ErbB signaling system in    development and disease. Adv Anat Embryol Cell Biol 190:1-65-   8. Buonanno A, Fischbach G D (2001) Neuregulin and ErbB receptor    signaling pathways in the nervous system. Curr Opin Neurobiol    11:287-296-   9. Buresova O, Krekule I, Zahalka A, Bures J (1985) On-demand    platform improves accuracy of the Morris water maze procedure. J    Neurosci Methods 15:63-72-   10. Costall B, Hui S C, Naylor R J (1978) Modulation of amphetamine    hyperactivity by DPI injected into rat nucleus accumbens    [proceedings]. Br J Pharmacol 64:461P-   11. Eilam R, Pinkas-Kramarski R, Ratzkin B J, Segal M, Yarden    Y (1998) Activity-dependent regulation of Neu differentiation    factor/neuregulin expression in rat brain. Proc Natl Acad Sci USA    95:1888-1893-   12. Esper R M, Pankonin M S, Loeb J A (2006) Neuregulins: versatile    growth and differentiation factors in nervous system development and    human disease. Brain Res Brain Res Rev 51:161-175-   13. Falquet L, Pagni M, Bucher P, Hulo N, Sigrist C J, Hofmann K,    Bairoch A (2002) The PROSITE database, its status in 2002. Nucleic    Acids Res 30:235-238-   14. Farmer A, Elkin A, McGuffin P (2007) The genetics of bipolar    affective disorder. Curr Opin Psychiatry 20:8-12-   15. Fischbach G D (2007) NRG1 and synaptic function in the CNS.    Neuron 54:495-497-   16. Geuna S, Nicolino S, Raimondo S, Gambarotta G, Battiston B, Tos    P, Perroteau I (2007) Nerve regeneration along bioengineered    scaffolds. Microsurgery 27:429-438-   17. Glabe C (2006) Biomedicine. Avoiding collateral damage in    Alzheimer's disease treatment. Science 314:602-603-   18. Go R C, Perry R T, Wiener H, Bassett S S, Blacker D, Devlin B,    Sweet R A (2005) Neuregulin-1 polymorphism in late onset Alzheimer's    disease families with psychoses. Am J Med Genet β Neuropsychiatr    Genet 139:28-32-   19. Golub M S, Germann S L, Lloyd K C (2004) Behavioral    characteristics of a nervous system-specific erbB4 knock-out mouse.    Behav Brain Res 153:159-170-   20. Guo W P, Wang J, Li R X, Peng Y W (2006) Neuroprotective effects    of neuregulin-1 in rat models of focal cerebral ischemia. Brain Res    1087:180-185-   21. Haddad P M, Sharma S G (2007) Adverse effects of atypical    antipsychotics: differential risk and clinical implications. CNS    Drugs 21:911-936-   22. Hahn C G, Wang H Y, Cho D S, Talbot K, Gur R E, Berrettini W H,    Bakshi K, Kamins J, Borgmann-Winter K E, Siegel S J, Gallop R J,    Arnold S E (2006) Altered neuregulin 1-erbB4 signaling contributes    to NMDA receptor hypofunction in schizophrenia. Nat Med 12:824-828-   23. Hanninen K, Katila H, Saarela M, Rontu R, Mattila K M, Fan M,    Hurme M, Lehtimaki T (2007) Interleukin-1 beta gene polymorphism and    its interactions with neuregulin-1 gene polymorphism are associated    with schizophrenia. Eur Arch Psychiatry Clin Neurosci-   24. Hoglinger G U, Breunig J J, Depboylu C, Rouaux C, Michel P P,    varez-Fischer D, Boutillier A L, Degregori J, Oertel W H, Rakic P,    Hirsch E C, Hunot S (2007) The pRb/E2F cell-cycle pathway mediates    cell death in Parkinson's disease. Proc Natl Acad Sci U S A    104:3585-3590-   25. Hoglinger G U, Rizk P, Muriel M P, Duyckaerts C, Oertel W H,    Caille I, Hirsch E C (2004) Dopamine depletion impairs precursor    cell proliferation in Parkinson disease. Nat Neurosci 7:726-735-   26. Holbro T, Hynes N E (2004) ErbB receptors: directing key    signaling networks throughout life. Annu Rev Pharmacol Toxicol    44:195-217-   27. Hong L E, Wonodi I, Stine O C, Mitchell B D, Thaker G K (2007)    Evidence of Missense Mutations on the Neuregulin 1 Gene Affecting    Function of Prepulse Inhibition. Biol Psychiatry-   28. Hu X, Hicks C W, He W, Wong P, Macklin W B, Trapp B D, Yan    R (2006) Bacel modulates myelination in the central and peripheral    nervous system. Nat Neurosci 9:1520-1525-   29. Karoutzou G, Emrich H M, Dietrich D E (2007) The    myelin-pathogenesis puzzle in schizophrenia: a literature review.    Mol Psychiatry-   30. Kwon O B, Longart M, Vullhorst D, Hoffman D A, Buonanno A (2005)    Neuregulin-1 reverses long-term potentiation at CA1 hippocampal    synapses. J Neurosci 25:9378-9383-   31. Li B, Woo R S, Mei L, Malinow R (2007) The neuregulin-1 receptor    erbB4 controls glutamatergic synapse maturation and plasticity.    Neuron 54:583-597-   32. Liberatore G T, Jackson-Lewis V, Vukosavic S, Mandir A S, Vila    M, McAuliffe W G, Dawson V L, Dawson T M, Przedborski S (1999)    Inducible nitric oxide synthase stimulates dopaminergic    neurodegeneration in the MPTP model of Parkinson disease. Nat Med    5:1403-1409-   33. Mathew S V, Law A J, Lipska B K, vila-Garcia M I, Zamora E D,    Mitkus S N, Vakkalanka R, Straub R E, Weinberger D R, Kleinman J E,    Hyde T M (2007) {alpha}7 nicotinic acetylcholine receptor mRNA    expression and binding in postmortem human brain are associated with    genetic variation in Neuregulin 1. Hum Mol Genet-   34. McIntosh A M, Moorhead T W, Job D, Lymer G K, Munoz M S, McKirdy    J, Sussmann J E, Baig B J, Bastin M E, Porteous D, Evans K L,    Johnstone E C, Lawrie S M, Hall J (2007) The effects of a neuregulin    1 variant on white matter density and integrity. Mol Psychiatry-   35. Meeks T W, Ropacki S A, Jeste D V (2006) The neurobiology of    neuropsychiatric syndromes in dementia. Curr Opin Psychiatry    19:581-586-   36. Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer    C, Kilger E, Neuenschwander A, Abramowski D, Frey P, Jaton A L,    Vigouret J M, Paganetti P, Walsh D M, Mathews P M, Ghiso J,    Staufenbiel M, Walker L C, Jucker M (2006) Exogenous induction of    cerebral beta-amyloidogenesis is governed by agent and host. Science    313:1781-1784-   37. Muller N, Schwarz M (2006) Schizophrenia as an    inflammation-mediated dysbalance of glutamatergic neurotransmission.    Neurotox Res 10:131-148-   38. Nadri C, Belmaker R H, Agam G (2007) Oxygen restriction of    neonate rats elevates neuregulin-1 alpha isoform levels: Possible    relationship to schizophrenia. Neurochem Int 51:447-450-   39. Owen M J, Craddock N, Jablensky A (2007) The genetic    deconstruction of psychosis. Schizophr Bull 33:905-911-   40. Ozaki M, Itoh K, Miyakawa Y, Kishida H, Hashikawa T (2004)    Protein processing and releases of neuregulin-1 are regulated in an    activity-dependent manner. J Neurochem 91:176-188-   41. Ozaki M, Sasner M, Yano R, Lu H S, Buonanno A (1997)    Neuregulin-beta induces expression of an NMDA-receptor subunit.    Nature 390:691-694-   42. Pertusa M, Morenilla-Palao C, Carteron C, Viana F, Cabedo    H (2007) Transciptional control of cholesterol biosynthesis in    Schwann cells by axonal neuregulin 1. J Biol Chem-   43. Przedborski S, Vila M (2003) The    1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model: a tool to    explore the pathogenesis of Parkinson's disease. Ann N Y Acad Sci    991:189-198-   44. Radde R, Bolmont T, Kaeser S A, Coomaraswamy J, Lindau D,    Stoltze L, Calhoun M E, Jaggi F, Wolburg H, Gengler S, Haass C,    Ghetti B, Czech C, Holscher C, Mathews P M, Jucker M (2006)    Abeta42-driven cerebral amyloidosis in transgenic mice reveals early    and robust pathology. EMBO Rep 7:940-946-   45. Rimer M, Barrett D W, Maldonado M A, Vock V M, Gonzalez-Lima    F (2005) Neuregulin-1 immunoglobulin-like domain mutant mice:    clozapine sensitivity and impaired latent inhibition. Neuroreport    16:271-275-   46. Ross C A, Margolis R L, Reading S A, Pletnikov M, Coyle J    T (2006) Neurobiology of schizophrenia. Neuron 52:139-153-   47. Schillo S, Pejovic V, Hunzinger C, Hansen T, Poznanovic S,    Kriegsmann J, Schmidt W J, Schrattenholz A (2005b) Integrative    proteomics: functional and molecular characterization of a    particular glutamate-related neuregulin isoform. J Proteome Res    4:900-908-   48. Schillo S, Pejovic V, Hunzinger C, Hansen T, Poznanovic S,    Kriegsmann J, Schmidt W J, Schrattenholz A (2005a) Integrative    proteomics: functional and molecular characterization of a    particular glutamate-related neuregulin isoform. J Proteome Res    4:900-908-   49. Schrattenholz A, Soskic V (2006) NMDA receptors are not alone:    dynamic regulation of NMDA receptor structure and function by    neuregulins and transient cholesterol-rich membrane domains leads to    disease-specific nuances of glutamate-signalling. Curr Top Med Chem    6:663-686-   50. Schubert C (2006) Alzheimer disease: BACE1 branches out. Nat Med    12:1123-   51. Scolnick E M, Petryshen T, Sklar P (2006) Schizophrenia: do the    genetics and neurobiology of neuregulin provide a pathogenesis    model? Hary Rev Psychiatry 14:64-77-   52. Thomson P A, Christoforou A, Morris S W, Adie E, Pickard B S,    Porteous D J, Muir W J, Blackwood D H, Evans K L (2007) Association    of Neuregulin 1 with schizophrenia and bipolar disorder in a second    cohort from the Scottish population. Mol Psychiatry 12:94-104-   53. Vila M, Przedborski S (2003) Targeting programmed cell death in    neurodegenerative diseases. Nat Rev Neurosci 4:365-375-   54. Willem M, Garratt A N, Novak B, Citron M, Kaufmann S, Ringer A,    Destrooper B, Saftig P, Birchmeier C, Haass C (2006) Control of    peripheral nerve myelination by the beta-secretase BACE1. Science    314:664-666-   55. Woo R S, Li X M, Tao Y, Carpenter-Hyland E, Huang YZ, Weber J,    Neiswender H, Dong X P, Wu J, Gassmann M, Lai C, Xiong W C, Gao T M,    Mei L (2007) Neuregulin-1 enhances depolarization-induced GABA    release. Neuron 54:599-610-   56. Xie F, Padival M, Siegel R E (2006) Association of PSD-95 with    ErbB4 facilitates neuregulin signaling in cerebellar granule neurons    in culture. J Neurochem-   57. Xu Z, Croslan D R, Harris A E, Ford G D, Ford B D (2005a)    Extended therapeutic window and functional recovery after    intraarterial administration of neuregulin-1 after focal ischemic    stroke. J Cereb Blood Flow Metab-   58. Xu Z, Croslan D R, Harris A E, Ford G D, Ford B D (2006)    Extended therapeutic window and functional recovery after    intraarterial administration of neuregulin-1 after focal ischemic    stroke. J Cereb Blood Flow Metab 26:527-535-   59. Xu Z, Ford G D, Croslan D R, Jiang J, Gates A, Allen R, Ford B D    (2005b) Neuroprotection by neuregulin-1 following focal stroke is    associated with the attenuation of ischemia-induced pro-inflammatory    and stress gene expression. Neurobiol Dis 19:461-470-   60. Xu Z, Jiang J, Ford G, Ford B D (2004) Neuregulin-1 is    neuroprotective and attenuates inflammatory responses induced by    ischemic stroke. Biochem Biophys Res Commun 322:440-446-   61. Yang X L, Huang Y Z, Xiong W C, Mei L (2005) Neuregulin-induced    expression of the acetylcholine receptor requires endocytosis of    ErbB receptors. Mol Cell Neurosci 28:335-346

1. A method of treating a subject suffering from Alzheimer's disease,comprising administering to said subject a recombinant soluble Type INeuregulin-1 β isoform which is an about 15 to about 35 kD N-terminalfragment of Type I Neuregulin-1β, wherein said recombinant soluble TypeI Neuregulin-1 β isoform is the first 245, 246, 247, 248, 249, or 250amino acids of Type I Neuregulin-1
 2. The method of claim 1, whereinsaid recombinant soluble Type I Neuregulin-1 β isoform is the first 245amino acids of Type I Neuregulin-1β.
 3. The method of claim 1, whereinsaid recombinant soluble Type I Neuregulin-1 β isoform crosses theblood-brain barrier.
 4. The method of claim 1, wherein said recombinantsoluble Type I Neuregulin-1 β isoform reduces dopaminergic cell death insaid subject compared to a subject that is not treated with saidrecombinant soluble Type I Neuregulin-1 β isoform.
 5. The method ofclaim 1, wherein the Type I Neuregulin-1 β isoform has an isoelectricpoint (p1) of about 4 to about
 10. 6. The method of claim 1, wherein theType I Neuregulin-1 β isoform is a modified polypeptide, wherein themodifications are selected from phosphorylation, glycosylation,methylation, myristoylation, oxidation and any combination thereof. 7.The method of claim 1, wherein said recombinant soluble Type INeuregulin-1 β isoform is administered in an amount of 3 to 600 ng/kgbody weight of the subject to be treated.
 8. The method of claim 1,wherein said recombinant soluble Type I Neuregulin-1 β isoform isadministered in an amount of 150 to 300 ng/kg body weight of the subjectto be treated.
 9. The method of claim 1, wherein said recombinantsoluble Type I Neuregulin-1 β isoform is administered in an amount ofabout 3, 5, 20, 50, 150, 200, 300 or 600 ng/kg body weight of thesubject to be treated.
 10. The method of claim 1, wherein saidrecombinant soluble Type I Neuregulin-1 β isoform is administered in anamount of about 20 ng/kg body weight of the subject to be treated. 11.The method of claim 1, wherein the administration is by intravenous orintraperitoneal injection.